DOE Bioenergy Technologies Office (BETO) 2015 Project Peer Review

Realization of Algae Potential Algae Biomass Yield Program

March 25, 2015 Technology Area Review

Peter Lammers, P.I. New Mexico State University -> Arizona State University

This presentation does not contain any proprietary, confidential, or otherwise restricted information Goal Statement • Develop an integrated process for producing 2,500 gallons of bio-fuel intermediate per acre per year through radical improvements in algal areal productivity and lipid content • Successful demonstration will advance the DOE goal of producing 5,000 gallons per acre per year by 2022 • Benefits to the U.S./BETO include – Increasing the viability and deployment of renewable energy technologies – Spurring the creation of a viable domestic bio-industry – Evaluation of engineered productivity enhancements in elite strain versus an extremophile strain chosen for crop rotation purposes – Evaluation of Sequential HTL options for nutrient recycling

2 Quad Chart Overview Timeline Barriers • March 1, 2014 • Barriers addressed • Sept 30, 2016 – Feedstock supply – Conversion R&D • % Complete 40% (time) – Sustainability

Budget Partners Total Costs as FY 15 Total Planned • Partners Costs of Costs Funding (FY o FY14- 12/31/14 15-Project End New Mexico State, Washington 15 Date (Non State and Arizona State Universities FFRDC) o Pan Pacific, Algenol Biofuels DOE $543K $4.265M o New Mexico Consortium Funded o National Labs: Argonne, Los NMSU/ASU $99.9K Alamos, Pacific Northwest Pan Pacific $83.2K o UOP-Honeywell (bio-crude NMC WSU $24.7K feedstock analysis) $0

*If there are multiple cost-share partners, separate rows should be used. 3 1 - Project Overview • Genetic improvements in mixotrophic algae - LANL/NMC – 40% enhancement from chlorophyll antenna size optimization – 3 fold improvement in oil content from cellobiose utilization • Horizontal photobioreactor cultivation - NMSU/ASU – EPA-exempt (40 CFR Part 725) outdoor testing of improved strains – Crop rotation for biological solution to temperature management – Calculate raceway productivity from climate simulation modeling • Sequential Hydrothermal Liquefaction - WSU/NMSU/ASU – Enables C, N and P recycle to cultivation – Cleaner fuel (less N) with bio-char used for HTL heat integration • Systems Modeling - Pan Pacific, Argonne, Algenol – LCA and TEA process models for iterative optimization of cultivation and pre-processing with mass and energy balance data – Modeling enables go/no-go decision for Phase 2 4 2 – Approach (Technical) • Key Challenges  Strain stability and thermo-tolerance via strain rotation

 Cultivation mixing system, CO2 delivery and energy consumption  Achieve harvest density of 2-3 g/L to lower harvesting costs  Heat integration and intermediate separations for SEQ-HTL  C, N and P recycle from HTL

5 2 – Approach (Management)

• Team includes all expertise required to execute the program  LANL/New Mexico Consortium – algal strain improvement  NMSU/ASU – outdoor cultivation and HTL testing  WSU – sequential hydrothermal liquefaction  PNNL – quantitative growth modeling  ANL – national leader in LCA  Pan Pacific – ASPEN modeling/TEA • Two face-to-face meetings (Jan. ‘14 & ’15) • Monthly team presentations via Adobe Connect • PI Visits to WSU (July ‘14) & LANL/NMC (Feb ‘15)

6

Aspen model of REAP unit operations Aspen model of PBR cultivation for REAP process has successfully been constructed in five stages:

• Stage 1- Pre-Reactor Components • Stage 2- Algal Growth • Stage 3- Downstream Processing • Stage 4- Model of SEQHTL unit operation • Stage 5- Linkage of stages for complete process model

Aspen model of PBR cultivation

Outcomes: • Integrated end-to-end Aspen system model for REAP process including CO2 absorption, simulations of algal growth reactors for PBR, downstream processing and SEQHTL processing.

• Initial fully converged Mass and Energy balances for integrated REAP process.

• Initial costing of process economics using Aspen database.

3 – Technical Accomplishments/ Progress/Results • Strain improvement - Chlorella

• Cultivation trials, PBR mixing systems and CO2 delivery

• Initial Sequential Hydrothermal Liquefaction Results

• TEA and LCA modeling

9 Strain Selection One elite strain, one extremophile 1. C. sorokiniana strain selection – UTEX 1230, 1228 and NAABB 1412 – Genomes are only ~5% conserved – Initial genetic toolkit developed for 1230 – Thermo-tolerance 1230 > 1412 > 1228 – Productivity 1412 > 1230 – Final selection for genetic enhancements is 1412 2. G. sulphuraria - summer season strain is a low-pH extremophile, tolerant to 56oC, low lipid/high carbohydrate, versatile heterotroph 3. Strain rotation for temperature management 10 REAP pairs this cultivation approach with highly synergistic sequential hydrothermal extraction technology (SEQHTL). This is a two-stage process that: i) extracts non-lipid fractions for recycling to oil productionObjectives,; ii) converts the remaining Outcomes lipid-enriched biom assand directly Impacts to cleaner bio-fue lby interme diate with lower nitrogen content; and iii) eliminates the need for unit operations involving drying or cellular disruption. Outdoor testing of enhancePriorityd strains in Alge Areanol’s inexp ensive and scalable PBRs provides Table 1. Summary of REAP objectives, outcomes, and success criteria Improved Algal Biomass Productivity – Priority Area 1 Objectives Outcomes Impacts 1. Genetic enhancement of mixotrophic 40% increase in biomass productivity 2500 gal/acre-yr by end of 2014 Chlorella sorokiniana and 300% increase in oil content 3500 gal/acre-yr by end of 2015 2. Enclosed PBR Cultivation Systems for Outdoor PBR systems for >3 gAFDW Productivity of Greater than 25 g/m2-day EPA-exempt Outdoor Testing Enhanced per Liter autotrophic density and at Lower CAPEX and OPEX vs. Strains; Raceway Productivity Derived 6 gAFDW per Liter mixotrophic density Harmonization Model (1) from Climate-Simulation Models >50% oil content by mass Improved Pre-processing Technologies – Priority Area 2 Objectives Outcomes Impacts 3. Innovative Harvesting and Dewatering Harvested algae resulting in 5% solids; Energy Expended Less than 10% of Systems dewatering resulting in 30% solids Energy Content direct input to SEQHTL 4. Sequential Hydrothermal Extraction Continuous SEQHTL process design Technologies to Produce Bio-Fuel balancing High Efficiency Bio-Oil Cleaner Bio-Fuel Intermediate qualified Intermediate with C, N and P Recycle to Extraction and Bio-char Production for for UOP Fuel Upgrade Specifications Cultivation Heating Energy Requirement Technical Advances to Enable the Integration of the Algal Biomass Unit Operations – Priority Area 3 Objectives Outcomes Impacts 5. Physical Integration and Systems Iteratively Optimized Unit Operations for Optimum End-To-End Productivity with Modeling Algal Cultivation and Preprocessing Mass and Energy Balance Data 6. Develop LCA, TEA and Process GREET, Techno-Economic and Aspen Go Decision Triggers Engineering and Model of Integrated Process Models Enabling go/no-go Decision for Design for 1 Acre Integrated Pilot (FEL- Phase 2 2) containment in line with standards administered by EPA TSCA (40 CFR Part 725) to allow outdoor cultivation of engineered strains. The PBR approach also allows higher harvest concentrations reducing energy demand. Biochar derived from liquefaction provides the heating source for SEQHTL, thus REAP produces no co-products and minimal waste. Indeed our entire process design was driven by the need for highly compatible unit operations allowing radical improvements in biomass and bio-oil yields. The REAP collaboration includes all expertise required to execute this program. NMSU, WSU, and Algenol have expertise spanning outdoor cultivation, harvesting, and fuel intermediate production. WSU was chosen for its sequential hydrothermal liquefaction (SEQHTL) technology for the advantages presented below. The LANL team is renowned for algal strain improvement and PNNL for its quantitative growth models that underpin resource assessment. Argonne National Laboratory has world- recognized strength in life cycle analysis. Algenol has integration experience and operational experience via their algae integrated biorefinery. Pan Pacific personnel have designed, deployed, and managed industrial scale biological processes for decades and have led earlier DOE algae R&D programs. Since Pan Pacific does not produce any algae technology, it was also chosen to provide checks and balances of the technologies being developed by the various team members. Thus, Pan Pacific leads the integration effort. REAP has engaged three industrial participants (Algenol, Pan Pacific and Phycal) with large roles, in part, to ensure a culture that makes things that can get out of the lab and into the field to satisfy the Algal Biomass Yield solicitation objective of making at least 2,500 gallons of bio-fuel intermediate per acre per year by 2018. The remainder of this proposal will elaborate upon the specific approaches REAP will take, the reasons for their selection, and the management plan that will achieve the REAP objectives on time.

4/1/2013 4 Progress towards antenna size optimization Not quite there yet

Strain Colonies Transgenics screened (Chl a/b ratios)

Cs-1228 300 2.0 Chl a/b ratio (2.0) Cs-1230 350 3.0 Chl a/b ratio (2.3)

Cs-1412 400 3.4 Chl a/b ratio (2.0) (optimum is 5)

The failure to achieve higher Chl a/b ratios in Cs-1228 and Cs-1412 transgenics may reflect DNA sequence dissimilarities with the Cs-1230 CAO RNAi gene (88% identity) or gene copy number (2X) Thermo-tolerance Issues Temperature data for 20 cm depth horizontal PBR.

The area between the red lines indicates acceptable temperature range for Galdieria

The area between the green lines shows temperature range in which more temperate algae like Chlorella would grow.

Sonora, Mexico Fort Myers, FL Maximum Specific Growth Rate as a Function of Temperature: Chlorella UTEX 1230R vs. DOE 1412

15 Enhanced heat tolerance in Chlorella sorokiniana – 1412 mutants over-accumulating (5X) proline 1228 1412 P51412

Growth at 40 oC • 1412 over-expressing 1-pyroline- 5-carboxylate synthase gene (P5CS) • P5CS transgenics have 20% greater productivity than the 35 5X 30 wild-type parent 1412. 25 20 15 per OD 750) OD per 10 1X Angela Pedroso Tonon, Free Proline (nmoles (nmoles Proline Free 5 Los Alamos National 0 12301228 1412 P51412 Laboratory Vampirovibrio chlorellavorous causing C. sorokiniana crashes? Results of PCR tests by Judy Brown @ Univ. Arizona Predatory Bacteria: Bdellovibrionaceae

Bdellovibrio

Epibiotic and periplasmic predation

Vampirovibrio chlorellavorus: • Obligate biotroph on Chlorella species • Gram-negative bacteria • 0.3 µm diameter-larger coccoid forms (0.6 µm) described (Coder and Star 1978) • Single, polar non-sheathed flagellum for motility 18 Jurkevitch 2007; Guerrero et al. 1986; A1- CecropinA Anti-Microbial Peptides tested for A2- CecropinB A3- Maginin2 inhibition of Vampirovibrio and A4- W16-CA A5- LactoferricinB A6- MsrA B1- Dermaseptin B2- Mellitin B3- TemporinA B4- TemporinF B5-TemporinL B6- TemporinG C1- Piscidin-1 C2- Piscidin-2 C3- Piscidin-3 C4- Empty well C5- Kanamycin (50ug/mL) C6- Carbenicillin (125mg/mL) D1- Uninfected Cs1412 D2- Uninfected Cs1412 D3- Empty well D4- Empty well D5- Cs1412 Infected with Vampiro Dr. Satish Rajamani D6- Cs1412 Infected with Vampiro Genetic Enhancements Schedule

20 Cultivation System Overview • Closed Plastic Photobioreactors – Mixing by paddlewheel – Mixing by hydraulic driven waterfoil (Algenol) – Hybrid tubular plastic with waterfoil Horizontal Photobioreactors Enclosed raceway design, passive solar heating, paddlewheel mixing, expandable from 4 to >75 linear feet

100 L/m2 at 10 cm depth

200 L/m2 at 20 cm depth Hybrid System

• Tubular Plastic Bag with hydraulic waterfoil mixing system • Easier to set up, avoids weakness at bulkhead fitting at ends of the Algenol bags (leak prevention) • Floated on a water basin to provide temperature control for summer growth of C. sorokiniana Rapid Molecular Diagnostics for Contaminant Detection

List of PCR primers:

Chlorophyta Rhodophyta Chlorellaceae Cyanidiaceae Galdieriaceae ~Product Coelastrella Chlorella sorokiniana C. caldarium/C. merolae G. sulphuraria Marker Primer set size (bp) NMSU1 1412 1230 5510 MS1-YNP 5572 5587.1 Rubisco Large subunit RbcL F/R 550 Euk. 18S rDNA Cdm F/R 750 Algal 18S rDNA ITS1 ITS 1F/2R 350-400 Chlorella specific 18S rDNA ChspeR/TresR 195 18S rDNA-ITS1 CdmF/ITS2R 1300 Chlorophyte plastid genes UCP5 F/R 1300 Rhodophyte plastid genes URP1 F/R 440-500 C. Merolae URA5.3 URA5F/6R 725 Cox2-Cox3 intergenic spacer Cox2f/3R 300-500 Simple Sequence Repeats SSR9 F/R 573 PCR product amplified Black box: universal (euk/algae) primer set; green box: green algae specific primer set; and red box: red algae specific No PCR product amplified primer set. Chlorella sorokiniana NAABB 1412.1 Peak Growth in enclosed horizontal PBR at 30 g/m2/day May 2014 in Las Cruces, NM

60 L 120 L 480 L Temp Spike - crash

5 60 4.5 50 4 3.5 40 3

2.5 30 OD 750 OD 750 OD 2 Temp ( C ) ( Temp Temp (C) 20 1.5 1 10 0.5 0 0 5/1/14 5/6/14 5/11/14 5/16/14 5/21/14 5/26/14 5/31/14 6/5/14 Date Biological Risk #2 Competitor Algae - Coelastrella

Electron micrograms - Peter Cooke

Strain isolation and characterization – Mark Seger 2013 Outdoor Growth of Galdieria sulphuraria CCMEE 5587.1** in an Enclosed Horizonal PBR Bag10 Grow cmth andDepth, Temperatu 5%re CO2 in Air, @ 2 L/min

3 55 Growth 2.5 Temp. 50

C) ° 2 45

1.5 40 y = 0.165x + 0.159

g (AFDW)/L 1 35

Temperature ( R² = 0.978 0.5 30 PEAK Growth 0 25 May/18/13 May/27/13 Jun/6/13 0.165 grams/L/day Date 16.5 grams/m2/day

** Retrospective DNA analysis documented this was actually a mixture of G. sulphuraria and C. merolae Effect of Sucrose on Outdoor Growth Rate of Galdieria sulphuraria 56 g/m2/day 6 60 25 mM 5 sucrose 50 200 L/m2 4 40

3 30 OD 750 OD Temp C Temp 2 20 OD 750 1 Bomono 10 OD 750 0 Sapphire 0 6/30/14 7/10/14 7/20/14 7/30/14 8/9/14 Date SeqHTL-Reaction Network

Bio-gas

Bio-oil Algae

WEs

Polysacch Algae arides Bio-char

Bio-gas

Stage 1 Stage 2 160C 240C CO2 Solar energy Pure Heterotrophic Growth of G. sulphuraria Microtiter plate assay DHTL- Coelastrella, Chlorella Nutrient Replete Cultures

90 80 Fig 5: HTL product distribution of Chlorella 70 60

50 40 Yield % 30 20 10 0 180 200 225 250 275 300 Biocrude Biochar WSC Gaseous products

Max Bio-oil yield: 14.75% Max Bio-oil yield: 33.6%

32 DHTL-C. merolae/G. suphuraria Nutrient Replete Cultures

Max Bio-oil yield: 16.98% Max Bio-oil yield: 21.22%

33 Results: Baseline Model

Emissions, gCO2e/MJ-RD REAP Davis, 2014 Chlorella 57 32 Galdieria 63 37

Must be below red line to be an advanced biofuel

. REAP has high emissions with the baseline assumptions . Cannot be overcome by increasing the productivity 34 Alternative Scenarios to reach 48 gCO2e/MJ

Scenario Chlorella (24 g/m2/d) Galdieria (15 g/m2/d)

Baseline 57 gCO2e/MJ-RD 63 gCO2e/MJ-RD Belt filter press 50 57 18 wt% feed to HTL 44 51 Use biochar for heat 36 42

. Biochar in 80% efficient boiler, 29 MJ/kg (HHV) - Recovery energy neglected

. Productivity must be kept above 12 g/m2/d to satisfy the emissions target - An underestimate!

35 Constraints on Experiments

Systerm Constraint Cultivation • Total energy < 48 KWh/ha/d • Likely need to be lower than this • If winter productivities are low, must reduce power further HTL • No solvents that require recycling? • Feed at 20 wt%? (under discussion) • Stage 2 yields measured with Stage 1 remnants from separation Harvest / dewater • Energy use less than that in current model • Cut costs . Quick and dirty TEA says we must solve these while cutting costs - Solve problems by avoiding them - Simplify the system 36 5 – Future Work • Issues: Staggered start FFRDCs funded one year ahead of Universities and Sub-contractors who worked “at risk” through calendar 2014 • Genetic enhancements are lagging behind schedule – Strain issues, construct regulatory elements • Low temperature water extractive reuse with Galdieria outdoors to enhance productivity to take advantage of high culture stability

• CO2 utilization in Algenol vs paddlewheel horizontal PBRs • Flocculant identification and dosing • SEQ-HTL design (focus on separations & heat integration) • Data collection with continuous flow HTL apparatus • Supply numerous data needs for TEA and LCA

37 Relevance/Key Objectives • Addresses many of BETO’s barriers

Aft-A Biomass Aft-B Sustainable algae Aft-C Biomass genetics availability production Aft-D Sustainable Aft-G Feedstock Aft-H Integration and harvesting properties scale up Aft-I Feedstock AFT-J Nutrient / material Tt-B Feed wet biomass processing recycle

• Contributes to specific MYPP milestones • By 2016, review integrated R&D approaches for high- yielding algal biofuel intermediates • By 2018, demonstrate 2500 gal/yr of biofuel intermediate (non-integrated unit processes)

38 Summary of Important Results

• Good agreement between maximum predicted and maximum observed productivity of wild type Chlorella sorokiniana 1412 (30 g/m2/day) • Range of HTL bio-crude oil yields 17-33% in nutrient replete conditions, batch mode • Galdieria cultivation is stable to carbohydrate addition for biomass productivity enhancement; wild-type readily uses cellobiose • 14 external conference presentations, 2 papers, 0 patents

39 Questions?

40 2014 Productivity - G. Sulphuraria 5587.1

• Tested productivity and HTL yields on a single G. sulphuraria and a single C. merolae strain among the Acidophilic Red Algae (Cyanidiales). • Lammers started a collaboration with Dr. Sherry Cady at PNNL who manages the Culture Collection of Microorganisms from Extreme Environments. • Plan to screen more Cyanidiales for important phenotypes: • Cell wall thickness or lack of cell walls (transformability, Andreas Weber) • Variant thermo-tolerance profiles CO2 Solar energy